Application of intestinal tract Ticam1 in regulation and control of MASH intestinal inflammation reaction and preparation of MASH medicine

Abstract

The invention provides an application of intestinal tract Ticam1 in regulation and control of MASH intestinal tract inflammatory response, the application is characterized in that the Ticam1 in the intestinal tract is inhibited, the growth of Ackermania mucilaginosa is promoted, and the strain code QAA37749.1 is used for catalyzing the conversion of betaine and improving MASH. The invention also provides an application of the intestinal tract Ticam1 in preparation of an MASH drug and a corresponding MASH treatment drug. Research finds that Ticam1 can aggravate intestinal injury of HFD mice, and deletion of Ticam1 in IEC can relieve HFD-induced hepatitis and hepatic fibrosis, improve HFD-induced metabolic disorder and cause change of intestinal microbiome, and further experiment and analysis are combined to obtain an influence mechanism of Ticam1 on MASH, so that a new thought and approach are provided for preparation of MASH treatment drugs, and the application has a broad application prospect. And the method has a remarkable application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of intestinal Ticam1 in regulating MASH intestinal inflammatory response and preparing MASH drugs. Background Art

[0002] Metabolic dysfunction-associated fatty liver disease (MASLD) is the hepatic manifestation of dysregulated lipid and glucose metabolism, often associated with obesity, dyslipidemia, and insulin resistance. Metabolic dysfunction-associated hepatitis (MASH) is considered to be a progressive form of MASLD, characterized by hepatic fat deposition, inflammation, hepatocellular damage, and varying degrees of fibrosis.

[0003] The gut-liver axis has a significant impact on MASLD. The "leaky gut" phenomenon exists in both MASLD patients and animal models, which can lead to the translocation of immune-active mediators, resulting in increased exposure to harmful substances in the liver, and then leading to liver inflammation and fibrosis. The immune response may damage the intestinal barrier. For example, IL-1β, lipopolysaccharide (LPS), and cytokines are all key mediators that damage the intestinal barrier. At the same time, diets that can induce the formation of MASLD are potent pro-inflammatory nutrients that trigger inflammatory cascades through inflammasome signaling pathways. For example, a high-fat diet causes a decrease in the proportion of Tregs and an increase in IL-17-producing γδT cells in the intestines of mice; a Western diet may cause dysbiosis and intestinal inflammation in aging mice; excessive fructose intake may lead to deterioration of the intestinal barrier, dysbiosis, low-grade intestinal inflammation, and endotoxemia.

[0004] Ticam1 is a protein containing a TIR domain that binds to Toll-like receptors (TLRs) through a "TIR:TIR domain" interaction, promoting signal transduction to initiate inflammatory responses against viral, bacterial, fungal, and parasitic pathogens. Ticam1 can be recruited directly by TLR3 or indirectly by TLR4 through the bridging adapter TRAM, which promotes the activation of NF-κB and MAPK signaling pathways and the formation of cytokines such as IL-1β and IL-18. However, the role of intestinal Ticam1 in MASH has not been reported. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an application of intestinal Ticam1 in regulating MASH intestinal inflammatory response and preparing MASH drugs, which inhibits Ticam1 in the intestine to promote the growth of Akkermansia muciniphila, which encodes QAA37749.1 to catalyze betaine conversion, thereby improving MASH.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] The invention discloses an application of intestinal Ticam1 in regulating MASH intestinal inflammatory response. The application is to promote the growth of Akkermansia muciniphila by inhibiting Ticam1 in the intestine. The bacterium encodes QAA37749.1, which catalyzes the conversion of betaine and improves MASH.

[0008] The invention discloses an application of intestinal Ticam1 in preparing a MASH drug. The application is to promote the growth of Akkermansia muciniphila by preparing an intestinal Ticam1 inhibitor. The bacterium encodes QAA37749.1, which catalyzes the conversion of betaine and improves MASH.

[0009] A drug for treating MASH is an intestinal Ticam1 inhibitor; the intestinal Ticam1 inhibitor can adopt an existing inhibitor with intestinal Ticam1 inhibitory function, for example, Pepinh-TRIF (TFA) (there is no research report related to MASH on this preparation), and further research and development can be carried out subsequently.

[0010] As one of the preferred embodiments of the present invention, the intestinal Ticam1 inhibitor treats MASH by inhibiting Ticam1 in the intestine.

[0011] As one of the preferred embodiments of the present invention, inhibiting the Ticam1 in the intestine can promote the growth of Akkermansia muciniphila, which encodes QAA37749.1 to catalyze the conversion of betaine and improve the MASH intestinal inflammatory response.

[0012] As one of the preferred embodiments of the present invention, the drug is in any pharmaceutical dosage form.

[0013] As one of the preferred embodiments of the present invention, the pharmaceutical dosage forms specifically include sugar-coated tablets, film-coated tablets, enteric-coated tablets, hard capsules, soft capsules, oral liquids, granules, pills, powders, injections, suppositories, ointments, plasters, creams, sprays, and drops.

[0014] The advantages of the present invention compared to the prior art are:

[0015] The present invention finds through research that Ticam1 can aggravate intestinal damage in HFD mice, while the lack of Ticam1 in IEC can alleviate HFD-induced hepatitis and liver fibrosis, improve HFD-induced metabolic disorders, and lead to changes in the intestinal microbiome. Further combined with experiments and analysis, the influence mechanism of Ticam1 on MASH is obtained, that is, by inhibiting Ticam1 in the intestine, the growth of Akkermansia muciniphila is promoted, and the bacterium encodes QAA37749.1 to catalyze betaine conversion and improve MASH. The present invention provides a new idea and approach for preparing MASH therapeutic drugs, and has a remarkable application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The results of Ticam1 exacerbating intestinal damage in HFD mice in Example 1 (Figure A shows the expression of Ticam1 in intestinal epithelial tissues of patients with and without MASH; Figure B shows the expression of Ticam1 in intestinal epithelial tissues of patients with and without MASH; fl / fl The difference in intestinal Ticam1 expression after mice were fed HFD and NCD; Figure C shows HFD-Ticam1 ΔIEC 、HFD-Ticam1 fl / fl 、NCD-Ticam1 ΔIEC and HFD-Ticam1 fl / fl The staining results of the ileum and colon of the four groups of mice; Figure D shows the FD4 flux results of each group of mice; Figure E shows the expression results of the intestinal barrier function protein ZO-1 in different groups of mice, with high green fluorescence levels; Figure F shows the expression results of the intestinal barrier function protein Occludin in the colon and ileum of different groups of mice; Figure G shows the expression results of intestinal inflammatory factor mRNA, namely MCP-1 and IL-1β, in different groups of mice; Figure H shows the inter-group differences in the levels of serum inflammatory factors in different groups of mice; and in the figure, * indicates p<0.05, ** indicates p<0.01);

[0017] Figure 2 The results of Example 1 in which Ticam1 deficiency in IECs alleviated HFD-induced hepatitis and liver fibrosis (Figure A is HFD-Ticam1 ΔIEC 、HFD-Ticam1 fl / fl 、NCD-Ticam1 ΔIEC and HFD-Ticam1 fl / flLiver tissue staining and statistical results of four groups of mice; Figure B shows the observation and statistical results of liver fibrosis in different groups of mice; Figure C shows the infiltration and statistical results of inflammatory macrophages in the liver of different groups of mice; Figure D shows the statistical results of liver weight in different groups of mice; Figure E shows the liver enzyme levels of different groups of mice, including ALT, AST, and ALP; Figure F shows the statistical results of the expression of inflammatory factors TNF-α, IL-1β and fat synthesis genes PPAR-γ and SREBP-1c in the liver of different groups of mice; and in the figure, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001);

[0018] Figure 3 The results of the improvement of HFD-induced metabolic disorders by the loss of Ticam1 in IECs in Example 1 (Figure A is HFD-Ticam1 ΔIEC 、HFD-Ticam1 fl / fl 、NCD-Ticam1 ΔIEC and HFD-Ticam1 fl / fl Figure A shows the volume and weight measurement and statistical results of mice in different groups; Figure B shows the statistical diagram of fat cells and diameters in white adipose tissue of mice in different groups; Figure C shows the glucose tolerance results of mice in different groups; Figure D shows the insulin tolerance of mice in different groups; Figure E shows the serum insulin level of mice in different groups; Figure F shows the level of HOMA-IR of mice in different groups; Figure G shows the 24-hour oxygen consumption monitoring and statistical results of mice in different groups; Figure H shows the 24-hour carbon dioxide production rate monitoring and statistical results of mice in different groups; Figure I shows the 24-hour heat production monitoring and statistical results of mice in different groups; and in the figure, * indicates p<0.05, ** indicates p<0.01, **** indicates p<0.0001);

[0019] Figure 4 The results of the intestinal microbiome changes caused by the loss of Ticam1 in IECs in Example 1 (Figure A is the PCA analysis result of the intestinal flora; Figure B is the HFD-Ticam1 ΔIEC 、HFD-Ticam1 fl / fl The relative abundance results of A. muciniphila, Alistipes shahii and Alistipes dispar in mice; Figure C shows the pictures of mice after the intestinal flora of the two groups of mice were eliminated by antibiotics; Figure D shows the statistical results of the weight and food intake of mice after the intestinal flora of the two groups of mice were eliminated by antibiotics; Figure E shows the MASH score of the mouse liver HE stain after the intestinal flora of the two groups of mice were eliminated by antibiotics, and Figure F shows the liver enzyme level of mice after the intestinal flora of the two groups of mice were eliminated by antibiotics; Figure G shows the difference in the expression of inflammatory factors and lipid synthesis genes in the liver of mice after the intestinal flora of the two groups of mice were eliminated by antibiotics; Figure H shows the results of glucose tolerance after the intestinal flora of the two groups of mice were eliminated by antibiotics; and in the figure, ** indicates p<0.01);

[0020] Figure 5 It is a diagram of the conversion result of betaine mediated by QAA37749.1 in A. muciniphila in Example 1 (Figure A is the quantitative statistical result of the differential metabolites and common metabolites in each comparison group; Figure B is a heat map of the relative contents of differential metabolites such as betaine in the two comparison groups; Figure C is the level of choline and betaine in the culture medium of wild-type E. coli BL21 and E. coli BL21 overexpressing QAA37749.1; Figure D is that the choline / betaine ratio of wild-type E. coli BL21 is significantly higher than that of E. coli BL21 overexpressing QAA37749.1; Figure E is an overall mechanism diagram; and in the figure, ** indicates p<0.01). DETAILED DESCRIPTION

[0021] The embodiments of the present invention are described in detail below. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the reagent products and experimental methods used in the following embodiments, unless otherwise specified, are conventional reagents or methods in the art and will not be repeated.

[0022] Example 1

[0023] This example is used to explore the mechanism of the effect of Ticam1 on MASH.

[0024] Toll-IL-1 receptor domain containing adaptor protein-1 (Ticam1) is a key molecule in the Toll-like receptor (TLRs) pathway. In this example, Ticam1 in mouse intestinal epithelial cells (IECs) was knocked out to inhibit the intestinal inflammatory response of metabolic dysfunction-associated steatohepatitis (MASH), and its mechanism of influence on MASH was studied.

[0025] The intestinal epithelial tissues of patients with MASH and patients without MASH (control) can be collected to compare the expression of Ticam1. ΔIEC ) mice and controls (Ticam1 fl / fl ) mice were fed a high-fat, high-fructose diet (HFD) or a normal chow diet (NCD) for 22 weeks, and the intestinal changes and MASH-related diseases of each group of mice were evaluated. Metagenomic sequencing was used to discover the changes in the intestinal microbiota between the different groups of mice. At the same time, liquid chromatography-mass spectrometry analysis was performed to evaluate the changes in serum metabolites caused by the intestinal microbiota.

[0026] The specific research process and results are as follows:

[0027] 1. Ticam1 exacerbates intestinal damage in HFD mice:

[0028] Intestinal epithelial tissues of patients with and without MASH were collected to compare the expression of Ticam1. The results showed that the expression of Ticam1 in the intestinal epithelial tissues of patients with MASH was higher than that of patients without MASH ( Figure 1 A), indicating that "Ticam1 in the intestinal epithelium is involved in the development of MASH".

[0029] Wild-type Ticam1 fl / fl The mice were fed a HFD or NCD diet for 22 weeks, and then their intestinal pathological changes were detected. The results showed that compared with mice fed a normal diet (NCD), mice fed a high-fat and high-fructose (HFD) diet had a significantly higher intestinal pathological changes in Ticam1. fl / fl In mice, the expression level of Ticam1 was increased, which is consistent with the results in humans ( Figure 1 B).

[0030] Evaluation of HFD-Ticam1 using FITC-dextran (FD4) ΔIEC 、HFD-Ticam1 fl / fl 、NCD-Ticam1 ΔIEC and HFD-Ticam1 fl / fl The intestinal permeability of the four groups of mice. The higher the intestinal permeability, the more likely it is that intestinal metabolites and some harmful substances enter the liver and systemic blood circulation through the portal vein, thus participating in the occurrence and development of the disease. HE staining results showed that there was no significant difference in the ileum and colon structure of the four groups, indicating that "gene knockout does not affect the basic structure of the intestine ( Figure 1 C)"; compared with HFD-Ticam1 fl / fl Mouse, HFD-Ticam1 ΔIEC The flux of FD4 from the intestinal lumen to the blood of mice was significantly reduced (p = 0.0387), indicating that "Ticam1 knockout improved intestinal permeability ( Figure 1 D)”; when fed with NCD, there was no significant difference between the two groups, indicating that “under normal diet, Ticam1 has no significant effect on intestinal permeability ( Figure 1 D)”.

[0031] Zonula occludens-1 (Zo-1) and occludin are both tight junction proteins, and their expression levels are positively correlated with intestinal mucosal barrier function. ΔIEC 、HFD-Ticam1 fl / fl 、NCD-Ticam1 ΔIEC and HFD-Ticam1 fl / flThe expression of Zonula occludens-1 (Zo-1) and occludin in the four groups of mice. The results showed that compared with HFD-Ticam1 fl / fl Mouse, HFD-Ticam1 ΔIEC Mice restored intestinal Zo-1 and occludin expression ( Figure 1 E~F).

[0032] Detection of HFD-Ticam1 ΔIEC 、HFD-Ticam1 fl / fl 、NCD-Ticam1 ΔIEC and HFD-Ticam1 fl / fl The expression of pro-inflammatory genes Mcp1 and IL-1β in the ileum of the four groups of mice. ΔIEC Improved ileal inflammation in HFD mice ( Figure 1 G), but the serum inflammatory factor detection did not seem to be significantly different between the groups ( Figure 1 H).

[0033] 2. Deletion of Ticam1 in IECs alleviates HFD-induced hepatitis and liver fibrosis:

[0034] The liver tissue was stained with HE and Sirius red. The results showed that HFD-Ticam1 fl / fl Mouse ratio, HFD-Ticam1 ΔIEC Less lipid accumulation in the liver, less hepatic necrosis, and less ballooning ( Figure 2 A), decreased fibrosis ( Figure 2 B); Histological analysis showed that knocking out Ticam1 in the intestinal epithelium could significantly improve liver damage.

[0035] F4 / 80 is a highly glycosylated G protein-coupled receptor that is used to identify mouse macrophages. Immunohistochemical staining of liver F4 / 80 showed that Ticam1 ΔIEC The number of macrophages in mice also decreased significantly ( Figure 2 C), indicating that the infiltration of inflammatory cells was reduced and the degree of inflammation was improved.

[0036] With HFD-Ticam1 fl / fl Mouse ratio, HFD-Ticam1 ΔIEC Mouse livers were lighter ( Figure 2 D), liver enzymes including alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels decreased significantly ( Figure 2 E), biochemical analysis showed that liver damage was significantly improved.

[0037] With HFD-Ticam1 fl / flMouse ratio, HFD-Ticam1 ΔIEC The gene expression levels of inflammatory factors IL-1β and TNF-a in mouse liver were significantly downregulated, and the fat synthesis genes PPAR-γ and SREBP-1c were also significantly downregulated ( Figure 2 F) Gene transcription level confirmed that intestinal Ticam1 knockout can improve liver inflammation and steatosis.

[0038] 3. Deletion of Ticam1 in IECs improves HFD-induced metabolic disorders:

[0039] After 22 weeks of HFD feeding, Ticam1 ΔIEC Mouse and Ticam1 fl / fl The mice ate no significant difference in their diet, but lost some weight ( Figure 3 A). White adipose tissue (iWAT) has smaller adipocytes ( Figure 3 B).

[0040] With Ticam1 ΔIEC Compared with control mice, Ticam1 fl / fl Mice had impaired glucose tolerance, increased fasting insulin levels, and increased insulin resistance index (HOME-IR) ( Figure 3 C, Figure 3 EF), but no significant difference was found in the insulin resistance test ( Figure 3 D).

[0041] Metabolic rate was then assessed using metabolic cages. fl / fl Compared with mice, Ticam1 ΔIEC Mouse whole body oxygen consumption rate ( Figure 3 G) and energy consumption rate ( Figure 3 I) increased, but the carbon dioxide production rate did not change significantly ( Figure 3 H). These results suggest that “intestinal Ticam1 knockout helps increase energy expenditure and improves metabolic disorders induced by HFD”.

[0042] The above three parts start from clinical phenomena, and by constructing high-fat-fed genetic mice, they test intestinal permeability, intestinal barrier function, intestinal inflammation level, liver inflammation, fatty changes and expression of related genes, as well as sugar metabolism, insulin levels and metabolic rate indicators, and finally make it clear that "knockout of Ticam1 in the intestinal epithelium can alleviate intestinal and liver damage in HFD mice and improve metabolic function". Next, we will further explore the specific mechanism by which knockout of intestinal Ticam1 improves the disease.

[0043] 4. Loss of Ticam1 in IECs leads to alterations in the intestinal microbiome:

[0044] Metabolic disorders are often associated with intestinal flora disorders. Metagenomic sequencing was used to detect the flora structure of the distal ileum and found that Ticam1 ΔIEC Mouse and Ticam1 fl / fl The microbiota structure of mice was significantly different ( Figure 4 A) Among them, Ticam1 ΔIEC Akkermansia muciniphila was significantly enriched in the mouse intestine, but Alistipes shahii and Alistipes dispar were significantly reduced ( Figure 4 B), in which Akkermansia muciniphila is a recognized probiotic, and its increase may help to improve HFD-induced metabolism-related fatty liver disease.

[0045] To determine whether the intestinal flora is responsive to HFD-Ticam1 ΔIEC Depletion of Ticam1 using broad-spectrum antibiotics plays a key role in improving disease in mice ΔIEC Mouse and Ticam1 fl / fl The original intestinal flora of mice was changed and high-fat diet was adopted. The weight and food intake of the two groups of mice ( Figure 4 CD), liver inflammation and fatty degeneration ( Figure 4 EF), liver inflammatory factors and the expression of fat synthesis genes ( Figure 4 G), glucose tolerance ( Figure 4 The above results indicate that "the changes in intestinal flora caused by Ticam1 knockout are crucial for disease improvement". Next, we will further explore how the increase in beneficial bacteria Akkermansiamuciniphila improves the disease.

[0046] 5. QAA37749.1-mediated betaine transformation in Akkermansia muciniphila:

[0047] Previous studies have shown that beneficial intestinal bacteria can metabolize and produce some beneficial small molecules to improve metabolic-related fatty liver disease. Therefore, non-targeted metabolomics was used to detect whether the serum metabolites of mice changed due to changes in the bacterial flora. The metabolomics results showed that the number of metabolites identified was Figure 5 As shown in A, by comparing the HFDWT group and the NCDWT group as well as the HFDKO and HFDWT mice and taking the intersection, 12 common metabolites were found, among which betaine was significantly decreased in HFDWT mice ( Figure 5 B), but significantly increased in NCDWT and Ticam1ΔIEC mice. Previous literature also showed that betaine plays an important role in anti-inflammation, improving insulin resistance and mitochondrial function.

[0048] VI. QAA37749.1 in Akkermansia muciniphila mediates betaine conversion:

[0049] Betaine in the body is mainly synthesized by microorganisms using choline, and the key step is the oxidation of choline to betaine aldehyde, which can be catalyzed by alcohol dehydrogenase. We hypothesized that Akkermansia muciniphila promotes the accumulation of betaine. By comparing the protease that catalyzes the conversion of choline to betaine with the gene-encoded protein of Akkermansia muciniphila, we found that a protein sequence had a high similarity with alcohol dehydrogenase. The gene is QAA37749.1 (existing known genes, for specific information, see https: / / www.ncbi.nlm.nih.gov / protein / QAA37749.1 / ). Subsequently, the gene was overexpressed in E. coli BL21 and cultured in a medium supplemented with choline ( Figure 5 C). Compared with wild-type E. coli BL21, the ratio of choline / betaine in the culture medium of E. coli BL21 overexpressing QAA37749.1 was lower ( Figure 5 D), indicating that the protein encoded by QAA37749.1 can promote the conversion of choline to betaine.

[0050] In summary, Ticam1 ΔIEC The intestinal damage of mice was alleviated and the degree of intestinal flora disorder was reduced. The abundance of Akkermansia muciniphila increased. The corresponding protein encoded by the QAA37749.1 gene of this bacterium can utilize choline and convert it into betaine, which helps to improve liver inflammation, fibrosis and metabolic disorders in HFD-fed mice.

[0051] Therefore, inhibition of Ticam1 in IECs may be a promising strategy for treating MASH-related diseases. Figure 5 E) For example, by preparing an intestinal Ticam1 inhibitor, the growth of Akkermansia muciniphila, which encodes QAA37749.1 and catalyzes betaine conversion, is promoted to improve MASH.

[0052] Example 2

[0053] A MASH therapeutic drug of this embodiment.

[0054] The drug uses Pepinh-TRIF (an intestinal Ticam1 inhibitor) as the main active ingredient, and is added with a pharmaceutically acceptable carrier as needed to prepare any pharmaceutically acceptable dosage form, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, hard capsules, soft capsules, oral liquids, granules, pills, powders, injections, suppositories, ointments, plasters, creams, sprays, and drops.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of intestinal Ticam1 in regulating MASH intestinal inflammatory response, characterized in that: The application is to promote the growth of Akkermansia muciniphila by inhibiting Ticam1 in the intestine, the bacterium encodes QAA37749.1, which catalyzes the conversion of betaine and improves MASH.

2. Application of intestinal Ticam1 in the preparation of MASH drugs, characterized in that: The application is to prepare an intestinal Ticam1 inhibitor to promote the growth of Akkermansia muciniphila, which encodes QAA37749.1 to catalyze the conversion of betaine and improve MASH.

3. A drug for treating MASH, characterized in that: It is an inhibitor of intestinal Ticam1.

4. The MASH therapeutic drug according to claim 3, characterized in that: The intestinal Ticam1 inhibitor treats MASH by inhibiting Ticam1 in the intestine.

5. The MASH therapeutic drug according to claim 4, characterized in that: Inhibiting Ticam1 in the intestine can promote the growth of Akkermansia muciniphila, which encodes QAA37749.1, catalyzing the conversion of betaine and improving the MASH intestinal inflammatory response.

6. The MASH therapeutic drug according to any one of claims 3 to 5, characterized in that: The medicament is in any pharmaceutical dosage form.

7. The MASH therapeutic drug according to claim 6, characterized in that: The pharmaceutical dosage forms specifically include sugar-coated tablets, film-coated tablets, enteric-coated tablets, hard capsules, soft capsules, oral liquids, granules, pills, powders, injections, suppositories, ointments, plasters, creams, sprays, and drops.

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